The goal of this proposal is to address at the molecular and cellular level the differential recognition of commensal and pathogenic bacteria by gingival epithelial cells. The oral epithelium is in direct contact with bacteria and plays an important role in the innate immune response by the expression of mediators and antimicrobial factors. Paradoxically, it is typically in a non-inflamed state despite constant exposure to bacteria. Consistent with this observation, Preliminary Data demonstrate that a pathogenic bacterium, P. gingivalis generally upregulates cytokine expression in oral epithelial cell cultures in contrast to a commensal bacteria, [unreadable] gordonii which does not. Mucosal epithelium in the gut is also able to maintain homeostasis while exposed to a high commensal bacterial load, yet retains the capacity to respond to pathogenic bacteria by upregulating an inflammatory response. The proposed studies will use liquid-air interface cultures of primary human gingival epithelial cells which form multi-layers of highly differentiated cells. Three representative bacteria will be examined: a well defined commensal, S. gordonii, an opportunistic commensal, F. nucleatum and a pathogenic bacteria, P. gingivalis. mRNA profiling will build upon Preliminary Data to establish whether this spectrum of bacteria exhibit significant differences in upregulating the innate immune response focusing on antibacterial factors including defensins, cytokines/chemokines and their receptors, pattern recognition receptors, intracellluar signalling molecules, and molecules that affect barrier function. Results will be verified for selected genes by real-time PCR and at the protein level. Aim 2 will establish whether each bacterium induces different signalling pathways in the epithelial culture system described for Aim 1. These studies will examine MAP kinase signalling and activation of transcription factors. Their functional importance will be assessed by use of siRNA or specific inhibitors. The second aspect of Aim 2 is to identify differential induction of signalling complexes by identifying molecules that associate with TLR2 on bacterial stimulation. The approach will use immunoaffinity minicolumns and mass-spectroscopy. Once identified, siRNA approaches will be used to investigate their functional significance. Aim 3 will utilize gnotobiotic mice to test the hypothesis that acquisition of a commensal bacteria at infancy attenuates the later response to pathogenic bacteria. The goal of this Aim is to investigate whether age-associated acquisition of a commensal flora induces bacterial tolerance.